| Powdery mildew caused by Blumeria graminis (DC.) E. O. Speer f. sp. tritici (Bgt) is aserious fungal disease of wheat worldwide and perennial occurred in the wheat growing areasof China. Deployment of resistant cultivars provides an effective approach for disease controlto eliminate the use of fungicides and minimize crop losses. Resistance gene diversity can berealized by searching for largely untapped of the rich genetic resources in wheat landraces andtheir wild relatives. The closely linked molecular markers with resistance gene can be used toquickly introgress the desired genes into commercial varieties or pyramiding differentresistance genes into a single genotype/cultivar for broad-spectrum resistance in wheatbreeding programs. Elucidating the molecular basis of plant disease resistance offers furtherpossibilities for durable resistance.The resistant Chinese wheat landrace (Triticum aestivum L.) Baihulu carrying the mlbhlgene and the common wheat lines N9134and N9738carrying the gene PmAS846introgressedfrom wild emmer accession As846(Triticum turgidum var. dicoccoides) were used toresistance spectrum analysis, molecular marker and comparative genomics analysis, in orderto map the powdery mildew resistance genes and get the closely linked markers formarker-assisted selection (MAS). Meanwhile, wheat microarray was used to investigate thetranscriptome patterns of N9134and possible defense processes regulated by PmAS846afterinoculation with Bgt isolate E09. The main results are included as follows:1. Chinese wheat landrace Baihulu confers a high level of resistance against a wide rangeof Bgt races, especially those currently prevailing physiological races in Shaanxi. Toinvestigate the inheritance of powdery mildew resistance and detect adjacent molecularmarkers, we constructed a segregating population of301F2plants and corresponding F3families derived from Baihulu/Shaanyou225. Genetic analysis revealed that a singledominant gene was responsible for seedling stage powdery mildew resistance in Baihulu. A genetic map comprising of Xgwm106, Xgwm337, Xgwm1675, Xgwm603, Xgwm789,Xbarc229, Xgpw4503, Xcfd72, Xcfd83, Xcfd59, Xcfd19and mlbhl spanned28.2cM onchromosome1D. All markers were inherited co-dominantly. Xgwm603/Xgwm789andXbarc229were flanking markers tightly linked to mlbhl at genetic distances of1.5and1.0cM,respectively. The mlbhl locus was located in chromosome bin1DS0.59-1.00delimited by theSSR markers Xgwm337and Xbarc229.Gene mlbhl was located in the same or a similar chromosomal region to Pm24. Whentested with a differential array of23Bgt isolates, Baihulu displayed a response pattern thatwas clearly distinguishable from that of Chiyacao and the varieties or lines possessingdocumented Pm genes. Allelism analysis indicated that it represented a new gene, eitherallelic or closely linked with Pm24or resided at a tightly linked locus in a gene cluster. Thenew gene was designated Pm24b.2. Gene PmAS846provides a potent resistance that is effective against21Chinese Bgtisolates with different virulence patterns. The common wheat line N9134and N9738arehighly resistant to Shaanxi prevailing races at both seedling and adult stages. Genetic analysisof two F2population and their F3families, developed from the cross of N9738/Huixianhongand N9134/Shaanyou225, indicated that N9134and N9738carrying one dominant resistancegene. A set of common wheat nullisomic (monosomic) lines were used to analyze thechromosomal location of PmAS846. The result revealed that PmAS846was located on wheatchromosome5B. Microsatellite markers on wheat chromosome5B were screened forpolymorphisms between parents and bulks. Genetic linkage map of PmAS846was constructedwith sixteen microsatellite markers. The two flanking markers Xgpw3191and XFCP1mappedPmAS846in chromosome bin5BL-0.75-0.76.3. The high level of collinearity exists between wheat chromosome5BL, Brachypodiumchromosome4and rice chromosome9. To develop additional markers, synteny betweenwheat, Brachypodium and rice was used to develop closely linked molecular markers toreduce the genetic interval around PmAS846. Twenty-six expressed sequence tag(EST)-derived markers were mapped to the PmAS846locus. A high resolution genetic mapfor PmAS846locus was constructed. The PmAS846locus was delineated to a0.8cM intervalflanked by the EST marker BG904722on the proximal side and the EST marker CJ840011onthe distal side. Five markers co-segregated with PmAS846in the F2population ofN9134/Shaanyou225. The markers order is conserved between wheat and Brachypodium, butrearrangements are present in rice. Two markers, BJ261635and CJ840011flanked PmAS846 and narrowed PmAS846to a region that is collinear with197kb and112kb genomic regionson Brachypodium chromosome4and rice chromosome9, respectively. The genes located onthe corresponding homologous regions in Brachypodium, rice and barley could be consideredfor further marker saturation and identification of potential candidate genes for PmAS846. Themarkers co-segregating with PmAS846provide a potential target site for positional cloning ofPmAS846.In order to evaluate the potential use of markers linked with PmAS846in MAS, theco-segregating or closely linked markers were validated on a number of resistant andsusceptible wheat genotypes. Specific resistant band types amplified by markers RG-36976,BJ261635, AL819406, RG-37900, FCP1and BI955376were only present in genotypescarrying PmAS846, but absent in other cultivars and lines without PmAS846, indicating thatthese markers particularly useful for MAS of PmAS846in wheat breeding programs.4. To ascertain the global framework of N9134gene expression during Bgt invasion, weanalyzed of55,052host genes interactions between wheat and Bgt. Combined histopathologyanalysis, Affymetrix wheat genome array was used to identify Bgt-induced genes, expressionpatterns and possible defense processes regulated by PmAS846in N9134. The significanceanalysis of microarrays (SAM) was used to identify transcripts that showed a differentiallyexpressed between Bgt inoculated and mock-inoculated. A total of2,408probe sets werefound to be more than2fold change expressed in at least one time point. To verify themicroarray data, quantitative RT-PCR time course analyses was carried out on21randomlyselected differentially expressed transcripts. The results of quantitative RT-PCR analysisconfirmed the reliability of the microarray data. To identify groups of genes with similarexpression patterns, a SOM analysis was carried out and the2,408probe sets were clusteredinto9groups. N9134exhibits both induction and repression of large sets of gene transcriptsduring Bgt infection. Among the2,408genes, there were homology of many genes linked topathogenesis-related genes, defense and stress response genes, signal transduction andtranscription factor genes involved in phenylpropanoid pathway, and resistance geneanalogues (RGAs). Genes encoding the pathogenesis related (PR) proteins, including theBeta-1,-3-glucanase, chitinases, protease inhibitor and thaumatin-like proteins were induced.5. Use of chromosome bin map of16,000ESTs loci and comparative genomics, a part ofdifferentially expressed genes related to disease resistant were mapped to wheat chromosomal5BL and the PmAS846interval. The probe set Ta.25929.1.A1_at, which was considered as acandidate disease resistant gene was located in the physical bin5BL14-0.75-0.76. The cDNA sequence of gene RGA-36976(Ta.25929) is2,543bp encoding a protein with721amino acids,and has a functional domain of NB-ACR and LRR. To characterize the relationship betweenRGA-36976and PmAS846, marker RG-36976was developed from introne sequencesinsertions/deletions (InDels) between resistant and susceptible parents. RG-36976was locatedon the proximal side of PmAS846in the genetic linkage map of the PmAS846. ThereforeRGA-36976was not the candidate genes but closely linked with PmAS846. |
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